The M2 proton channel protein of the influenza A virus is the target of the anti-influenza drug, amantadine. M2 contains a single transmembrane domain that forms the homo-tetrameric pore of this channel. M2's small size and simple structure makes it an attractive model for understanding the mechanism of charge-stabilization and proton conduction through membrane proteins. In the previous period, high-resolution NMR and crystal structures were solved to elucidate the mechanism of proton conduction. The channel has a long, water-filled pore that leads to a selectivity filter defined by His37 and Trp41 Protons diffuse through this aqueous pore to bind at the His37 tetrad and open the Trp41 gate. To understand the structural basis for this process, structures will be solved in the next funding period. Influenza A virus is a major threat to human health. There are two different classes of approved anti-influenza drugs: amantadine and rimantadine target the M2 proton channel, while Tamiflu (oseltamivir) and related compounds target neuraminidase. Resistance to both classes of drugs poses a major problem, and most isolates of influenza A virus are now amantadine-resistant. We therefore are solving structures of amantadine and rimantadine complexes with M2, and drug-resistant mutants. Drug binds to M2 by targeting its ammonium to one of the three low energy hotspot sites aligned along the channel axis. Only a handful of mutations are tolerated at these sites in transmissible viruses. Here, hypothesis-directed and structure-based approaches are used to design new inhibitors of this set of resistant mutants. This endeavor will not only provide new leads for anti-influenza medications, but it should also advance structure-based design of drugs targeting membrane proteins - an increasingly important endeavor as the number of medicinally important membrane protein structures grows. In Aim 1, hypothesis-directed structural approaches are used to discover small molecules that inhibit relevant mutants of M2. In Aim 2, EPR, crystallographic, and NMR investigations will probe the mechanism of conduction and drug inhibition. Site-directed spin labeling will focus on the full-length protein in phospholipid vesicles, and evaluate conformational changes due to variations in pH and drug-binding. These studies will facilitate understanding of more high-resolution crystal structures and NMR structures. Crystallographic structures of M2 mutants will be solved with and without bound drugs to inform drug design in Aim 1 and also probe the mechanism of proton conduction. Our early crystallographic work was conducted with the isolated transmembrane domain in micelles; the membrane environment as well as missing domains might influence the structure. Thus, we are now crystallizing longer constructs from both bicelles and lipidic cubic phases. To help stabilize these constructs in their native conformations, we are solving structures with known therapeutic antibodies and antibodies selected on phage. Finally we will structurally characterize the proton channel of influenza B virus, BM2, which shows no sequence similarity to M2 aside from having a His-X3-Trp motif. These studies will also enable future structure-based drug design of BM2 inhibitors. In parallel we will conduct solution NMR studies in micelles, bicelles, and nanodisks. We combine biosynthetic and synthetic labeling strategies to facilitate structure determination and to increase the resolution of solution NMR structures. These studies will provide new insight into the mechanism of proton conduction through M2 and lay the groundwork for the design of new inhibitors.